Hematopoiesis is the development and formation of blood cells in the bone marrow, and is critical to the proper functioning of the immune response. Differentiation of the myeloid cell lineage (granulocytes and monocytes/macrophages) termed myelopoiesis commences in the human fetus at approximately six weeks of gestation. In the early stages of myelopoiesis, colony-forming units for granulocytes/monocytes (CFU-GMs) can be induced along either the granulocyte or monocyte pathways. Induction of the CFU-GM's along the granulocyte pathway results in distinct morphological stages of development, ultimately terminating in the characteristic trilobed structure of polymorphonuclear leukocytes, also known as granulocytes.
Induction of CFU-GMs along the monocyte pathway gives rise initially to proliferating monoblasts. Monoblasts differentiate into promonocytes and, ultimately, into mature monocytes. Monocytes are considered to be circulating immature macrophages, which are highly differentiated cells found in various tissues.
Monocyte-macrophages are known to secrete a number of biologically active polypeptides called cytokines that affect the functions of other cells. Interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) are all cytokines secreted by monocytes/macrophages that play an important role in hematopoiesis.
A continued need exists for the further identification and characterization of the other cytokines and growth factors involved in hematopoiesis and immunoregulation.
Abnormal expression of the genes encoding the various cytokines and growth factors involved in cell differentiation and proliferation can result in neoplasias, including leukemias. Leukemia is defined as a progressive malignant disease of the blood-forming organs, characterized by distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. The leukemias account for approximately 3 percent of all cancers in the United States. (Li, F. P., “The Chronic Leukemias: Etiology and Epidemiology,” in Neoplastic Diseases of the Blood, vol. I, pp. 7–17, Wiernik et al. eds. (1985)).
Oncogenes have been implicated as a cause of human leukemias. Gelmann, E. P. et al., “The Etiology of Acute Leukemia: Molecular Genetics and Viral Oncology,” in Neoplastic Diseases of the Blood, vol. I, pp. 161–182, Wiernik et al. eds. (1985). An oncogene is a gene that brings about or contributes to neoplastic transformation of cells by encoding proteins which regulate cell growth and differentiation. Retroviral and cellular oncogenes arise from cellular genes called proto-oncogenes, which appear to play an important role in normal hematopoietic cell growth and differentiation.
The isolation and characterization of viral oncogenes (v-onc) have facilitated the cloning and identification of the cellular oncogenes (c-onc) which derive their names from the respective viral genes. They are highly conserved among species, and homologs are found in all vertebrates, in lower organisms, and in humans. (Gelmann et al.) The role of c-onc genes in neoplasia has been investigated extensively.
The retroviral oncogene v-myb transforms myelomonocytic hematopoietic cells in vivo and in vitro. (Moscovici, C. et al., Adv. Viral Oncol. 1:83–106 (1982)). The v-myb oncogene was originally defined by two naturally occurring avian retroviruses, AMV and E26, that induce myeloid leukemias in chickens. (Moscovici et al.) The v-myb oncogenes are derived from a normal, cellular proto-oncogene, c-myb, which is expressed in high levels in all immature hematopoietic lineages. (Klempnauer, K. H. et al., Cell 31:537–547 (1984)). In contrast, v-myb oncogenes only transform a few cell types, such as the immature myeloid precursors of neutrophils and macrophages. Both c-myb and v-myb encode nuclear, DNA binding proteins (i.e. transcription activators) that regulate the phenotypes of normal and transformed hematopoietic cells respectively. (Ness, S. A. et al., Cell 59: 1115–1125 (1989); Burk, O. and Klempnauer, K. H., EMBO J. 10(12):3713–3719 (1991)). The transforming activity of these proteins is regulated by cell type-specific cofactors. The DNA-binding domain of the v-myb proteins corresponds to the domain of several other myb-related DNA-binding proteins isolated from such diverse species as mammals, insects, and plants. (Queva et al. 1992)
An interesting feature of the v-myb oncogene is that it not only blocks differentiation, but it also dictates the differentiation phenotype of the myeloid cells that it transforms. (Ness, S. A. et al., Cell 59:1115–1125 (1989)). Expression of v-myb in myeloid cells results in them acquiring an immature phenotype. (Burk and Klempnauer, 1991). In addition, it has been shown that minor changes in the structure of the v-Myb protein determine whether the transformed cells take on the phenotype of immature macrophages or immature granulocytes, (Golay, J. et al., Cell 55:1147–1158 (1988)). Moreover, temperature-sensitive v-myb transformed cells induced to differentiate can be induced to retrodifferentiate. (Introna, M. et al., Cell 63:1287–1297 (1990)). Different forms of v-myb impose alternate phenotypes of differentiation on transformed myeloid cells by regulating unique sets of differentiation specific genes. (Introna, M. et al., Cell 63:1287–1297 (1990)).
Two genes, identified as mim-1 and MD-1, are known to be regulated by v-myb. (Ness et al., 1989; Burk and Klempnauer, 1991). The mim-1 gene is specifically expressed in normal, immature, granulocytes and encodes a 35 kD secretable protein that is stored in the granules of those cells. (Ness et al., 1989; Queva, C. et al., Development 114:125–133 (1992)). Indeed, mim-1 encodes one of the most abundant proteins found in granulocytes, and the high level of expression suggests that it may be a structural component of the promyelocyte granule. (Ness et al. 1989). When promyelocytes undergo terminal differentiation to neutrophil granulocytes, a decrease in the level of mim-1 protein is observed. (Queva et al.) Moreover, analysis of chick embryo development shows that mim-1 mRNA transcripts are found where granulopoiesis occurs. (Queva et al.) Thus, mim-1 is the first described marker for cells that are differentiating into the granulocytic lineage. (Queva et al.; Introna et al.).
The mim-1 gene is one of a number v-myb-regulated genes that contribute to the unique differentiation phenotype displayed by both normal and transformed myeloid cells. Those genes, which include MD-1, must by definition be regulated similarly to mim-1 by the various forms of the v-myb protein. (Ness et al.) It is likely that a number of different structural changes to the myb protein will alter the phenotype of myeloid cells transformed by the v-myb oncogene and affect its capacity to regulate its target genes, including mim-1 and MD-1. (Introna et al.)
Thus, v-myb acts as a master gene in hematopoietic cell differentiation by regulating the expression of a unique set of genes within the myelomonocytic lineage. (Introna et al.) Because these genes are expected to be important regulators of cell differentiation and proliferation, their identification is critical to understanding the molecular mechanisms of neoplasia, transformation, and growth control. Thus, a need exists in the art for the identification of other genes involved in hematopoietic cell differentiation.